Project Abstract Neoplasms of the choroid plexus (CP) are rare primary brain tumors predominantly found in childhood. CP tumors range from the more common and benign CP papilloma (CPP), to the rare CP carcinoma (CPC) that is poorly understood but highly lethal. Treatments for CPC include surgery, radiation, and chemotherapy that cause severe long term side effects in survivors. Development of more effective and less debilitating therapies for CPC has been hampered by limited knowledge of the role of specific molecular defects, including abnormal NOTCH signaling and recurrent genomic alterations, in CPC. Insights into how these genes and pathways affect proliferation and growth of CPC will lead to targets for new therapies that can specifically suppress tumor growth without deleterious effects on the developing brain. By inducing sustained NOTCH1 expression, we developed mouse models of CP tumor that recapitulate CPP in humans. Lineage studies revealed that NOTCH-induced CPP originates from roof plate progenitors, both of which exhibit active NOTCH signaling and undergo proliferation driven by Sonic Hedgehog (SHH) from CP epithelium. In contrast, CP epithelial cells with clustered multiple primary cilia on the apical surface fail to respond to SHH, suggesting that the NOTCH pathway suppresses multi-ciliogenesis to preserve the singular primary cilium critical for the SHH signaling. Though key genes of the multi-ciliation network driven by Geminin coiled-coil domain-containing protein 1 (GEMC1) are expressed in CP epithelium, the transcriptional program is suppressed in NOTCH-activated CPP that lacks multi-ciliated cells. Conversely, GEMC1 loss results in the lack of multi-ciliation in the CP. In Aim 1, we will investigate GEMC1-directed multi-ciliate differentiation during CP development and tumorigenesis. We will determine whether activation of GEMC1 transcriptional cascade is sufficient to overcome constitutive NOTCH signaling to induce multi-ciliogenesis, and attenuate SHH signaling in CP tumor. Functional studies of GEMC1 and identification of its transcriptional targets will establish the molecular mechanisms of multi-ciliation in the CP, and uncover potential therapeutic venues for CP tumor. Consistent with abnormal SHH and NOTCH signaling in CP tumor in humans, simultaneous activation of both pathways in mice leads to malignant CP tumors that exhibit solitary primary cilium. Similarly, CPCs in humans are characterized by singular primary cilium and recurrent genomic alterations affecting crucial regulators of multi-ciliate differentiation. In Aim 2, we will utilize novel mouse models to investigate the molecular mechanisms of the genetic interaction between the NOTCH and SHH pathways and evaluate potential targeted therapeutics. We will determine whether the loss of GEMC1 drives CPC in collaboration with constitutive SHH signaling. The proposed studies will significantly advance our understanding of CPC and identify potential therapeutic targets.